The first use of enzyme-labeled antibodies in immunocytochemical detection was reported in 1966 by Avrameas and Uriel [C. R. Seances Acad. Sci. Ser., D262, 2543 (1966)] and Nakane and Pierce [J. Histochem. Cytochem., Volume 14:929 (1966)]. The importance of these enzyme-labeled antibodies in diagnostic medicine was not understood accurately until about 1971, when Engvall and Perlmann [Immunochemistry, Volume. 8, 871 (1971)] and van Weeman and Schuurs [U.S. Pat. No. 3,654,090 (1972)] described the use of antibody-enzyme conjugates in quantitative enzyme-linked immunoassays. Since that time, there have been numerous publications describing procedures for coupling antibodies and their fragments to various enzymes.
A class of heterogeneous immunoassay which is achieving widespread use and which employs an enzyme-labeled antibody is referred to as an immunometric assay. One type of immunometric assay is the so-called sandwich assay. In a sandwich assay, a ternary complex is formed from (i) a solid-phase, unlabeled antibody, (ii) the analyte of interest, and (iii) a soluble, labeled antibody. Sandwich assays require the compound of interest to be polyvalent, i.e. to have two or more different antigenic determinants or a single, multiply occurring determinant.
Sandwich assays can be divided into forward, reverse, and simultaneous assays, depending on the method of addition of the labeled antibody. In a forward sandwich assay, the labeled antibody is added to a preformed binary complex of solid-phase antibody and analyte. In a reverse sandwich assay, labeled antibody and analyte are allowed to complex before addition of the solid-phase antibody. In a simultaneous sandwich, the sample containing analyte is contacted simultaneously with both the labeled and the solid-phase antibodies. Forward assays require two washing steps, while reverse and simultaneous assays require only a single washing step.
A single antibody immunometric assay, first described by Schuurs and van Weeman [U.S. Pat. No. 3,654,090 (1972)] and extended by Mochida and Ogawa [U.S. Pat. No. 4,200,436 (1980)] and Freytag et al. [Clinical Chem., Volume 84, 417 (1984)] adds elements of simplicity, speed, versatility, and overall sensitivity. Furthermore, the analyte need not be polyvalent. In this assay format, an excess of labeled antibody (preferably monovalent) is mixed with a sample containing analyte. After a brief incubation period to allow binding of the analyte, excess unreacted labeled antibody is removed by exposure of the mixture to antigen which has been immobilized on a solid support. The free (or bound) fraction is then quantified and is a direct measure of the analyte concentration.
It has been shown that the quality and nature of the antibody-enzyme conjugate has profound influence on the ultimate usefulness of an immunoassay for the quantitation of antigens. Ishikawa et al. report that monomeric non-aggregated Fab'/enzyme conjugates yield the lowest non-specific background and the highest sensitivity, particularly in sandwich-type double antibody immunometric assays [Immunoenzymatic Techniques, ed. S. Avrameas, Elsevier Science Publ. B.V. pp. 219-232 (1983)]. Monomeric Fab-enzyme conjugates are also necessary for maximum performance in single antibody immunometric assays [Ishikawa, cited above, and co-pending patent application (IP-0458)]. However, when very low levels of analyte are being analyzed (&lt;1 fmole), these assays require extended incubation periods for the generation of significant quantities of enzymic product for measurement.
Covalent methods for coupling single enzyme molecules to single coupling sites on antibodies are known, e.g. Imagawa et al., J. Appl. Biochem, Volume 4, 400 (1982).
It has been appreciated for some time now that aggregated antibody-enzyme conjugates can provide enhanced signal generation over that of purely monomeric antibody-enzyme conjugates. Aggregated antibody-enzyme conjugates generally have been prepared using nonspecific coupling chemistries such as glutaraldehyde crosslinking [Engvall & Perlmann, Immunochemistry, Volume 8, 871 (1971)] or periodate oxidation [Boorsma & Streefkerk, J. Immunol. Methods, Volume 30, 245 (1979)]. The preparation of these conjugates involves a random crosslinking process in which the antibody or antibody-fragments can be buried deep within an aggregated complex, thus remaining largely inaccessible for antigen binding. In addition, these amorphous macromolecular complexes are difficult to prepare in a reproducible fashion. Immunoassays utilizing such aggregated antibody-enzyme conjugates routinely suffer from very high background blanks due to nonspecific binding, and the ultimate sensitivity achievable in immunometric assays is dramatically reduced [Ishikawa et al., Ann. Clin. Biochem., Volume 19, 379 (1982)].
Leary et al. report the formation of polymerized alkaline phosphatase by crosslinking the monomeric enzyme with disuccinimidyl suberate. The polymeric enzyme was then biotinylated with biotinyl-.epsilon.-aminocaproic acid N-hydroxysuccinimide ester. The biotinylated enzyme was then reacted with an excess of avidin. The resulting conjugate was then reacted with biotinylated nucleic acid probes leading to a noncovalent linkage of polymerized enzyme to probe. [Leary et al., P.N.A.S. (U.S.A.), Volume 80, 4045 (1983).]
A number of procedures have been reported that utilize noncovalent chemistry to generate antibody-enzyme conjugates with high enzyme-to-antibody ratios. For example, Butler describes an antibody-enzyme conjugate comprising immune complexes of peroxidase-antiperoxidase antibody or phosphatase-antiphosphatase antibody. [Butler, Methods Enzymol., Volume 73:482-523 (1981)]. Halbeck and Nepom describe an antibody-enzyme conjugate comprising complexes of protein A and anti-protein A-horseradish peroxidase conjugates. [Holbeck & Nepom, J. Immunol. Methods, Volume, 60, 47 (1983)]. Guesdon et al. describe a procedure for preparing antibody-enzyme complexes using conjugates of bovine serum albumin and enzyme labeled anti-bovine serum albumin antibody. [Guesdon et al., J. Immunol. Methods, Volume 58, 133 (1983)]. Finally, Yolken et al., describe antibody-enzyme conjugates comprising complexes of avidin and enzyme-labeled biotin. [Yolken et al., J. Immunol. Methods, Volume 56, 319 (1983)]. The aforementioned procedures all suffer from the disadvantage that the linkage of antibody to enzyme is not covalent, and, therefore, a reversible binding results which is susceptible of unwanted dissociation.
There is a need for a highly reproducible procedure for the preparation of convalently linked polymeric-enzyme/antibody conjugates in which both original enzymatic activity as well as original immunoreactivity are maintained.